Effects of the ph on growth and morphology of Anabaenopsis elenkinii

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1 Fottea 11(1): , Effects of the ph on growth and morphology of Anabaenopsis elenkinii Mi l l e r (Cyanobacteria) isolated from the alkaline shallow lake of the Brazilian Pantanal Kleber Renan de Souza Sa n t o s, Fernanda Rios Ja c i n a v i c i u s & Célia Leite Sa n t Anna Laboratory of Phycology, Institute of Botany, Av. Miguel Estéfano 3687, CEP , São Paulo SP, Brazil; e mail: santoskrs@gmail.com Abstract: Anabaenopsis elenkinii Mi l l e r forms bloom in the alkaline shallow lakes of the Brazilian Pantanal. The A. elenkinii CCIBT1059 strain was isolated from one of these alkaline lakes and the experiments were made in growth chamber during 30 days, under modified medium BG 11 (3 % NaNO 3 ), temperature 25 ºC, photoperiod light dark cycle and irradiance of µmol photons m 2 s 1 at three different ph values: 7.0, 9.5 and In relation to growth rate and cell yield the higher values were observed at ph Morphologically, the longest trichomes were found at ph 7 (maximum 45 cells) in comparison with ph 9.5 (maximum 32 cells) and ph 10.5 (maximum 23 cells). The occurrence of heterocytes was observed in all treatments, but akinetes were never formed. The morphometric variability within each treatment between exponential and stationary growth was clearly higher than the variability among different treatments in most cases. Our results indicate that A. elenkinii is typical of alkaline systems and also that in lower ph values the growth limitation can occur in terms of number of cells and biomass. This study represents the first experimental evidence of the effects of ph on growth rate, cell yield and morphometric variability of A. elenkinii. Key words: alkaline lakes, Anabaenopsis elenkinii, Brazilian Pantanal, Cyanobacteria, ph, wetland Introduction The Pantanal is a large wetland of about 160,000 km² located in the center of the South American continent (Ju n k et al. 2006). The Brazilian Pantanal is divided into eleven sub regions that are characterized by the relief, soil, vegetation and flood pulse (Si l v a & Ab d o n 1998). The studied sub region, Pantanal of Nhecolândia, displays hundreds of typical and exclusive shallow lakes with high ph values (>9). Therefore, these lakes can be considered as extreme environments for most life forms (Kr i s t j á n s s o n & Hr e g g v i d s s o n 1995). These alkaline lakes were isolated from rivers or streams and the main source of water is rainfall and groundwater (Ba r b i é r o et al. 2002). In the alkaline lakes there are no macrophytes or fish and the dominant organisms are cyanobacteria (Santos & Sant Anna 2010). The high values of ph, electric conductivity, nutrients concentrations, cyanobacteria density and the lack of fish in the alkaline lakes from Pantanal of Nhecolândia are comparable to those of shallow lakes from arid regions of Australia (Hart et al. 1991), Nigeria (Egborge 1994), Ethiopia (Tudorancea & Ha r r i n s o n 1988) and Africa (Ballot et al. 2008). In these Brazilian extreme lakes, Anabaenopsis elenkinii Miller is the dominant organism and forms blooms (Fig. 1 a,b), mainly in the dry season (Santos & Sant Anna 2010). In spite of being the dominant species in the alkaline lakes from the Pantanal and potentially toxic (Carvalho et al. 2008), A. elenkinii is still little known in terms of development. Thus, our objective is to investigate the effects of ph on growth and morphology of A. elenkinii CCIBT1059, isolated from Brazilian Pantanal.

2 120 Santos et al.: Growth and morphology of Anabaenopsis elenkinii Table 1. Growth rate, duplication time and cell yield of the Anabaenopsis elenkinii strain CCIBT1059, in the exponential growth at different treatment of ph. The cultures were incubated at 25ºC, irradiance of µmol photons.m 2.s 1 on a hr light dark cycle on modified medium BG 11 (3% NaNO 3 ). Different letters after the date indicate significant differences at p < 0.05 (n = 3). Exponential growth (day) Growth rate (µ; day -1 ) Duplication time (G; day -1 ) Cell yield (R; cells.ml -1 ) ph to 8 0,259 a 2,68 a a ph to 4 0,261 a 2,67 a a ph to 7 0,349 b 1,99 b b Materials and Methods The studied nonaxenic strain CCIBT1059 is kept in modified liquid medium BG 11 (3% NaNO 3, indicated as BG 11m), ph 9.5, temperature 23 ± 1 ºC, photoperiod light dark cycle and irradiance of µmol photons.m 2.s 1 (provided by common day light fluorescent lamps and measured with a quanta meter sensor Li COR model LI 250 spherical) in the Cyanobacteria Culture Collection of Institute of Botany, São Paulo, Brazil. If was isolated from the alkaline shallow lake named Salina do Meio ( S and W ), State of Mato Grosso do Sul, Brazilian Pantanal. The morphological and metric (n = 200) characterization of material from nature and from culture were done under light microscope (Zeiss Axioplan 2). The classification system of Ho f f m a n n et. al. (2005) and taxonomic review of Ko m á r e k (2005) were adopted. At the end of the exponential phase, one aliquot of the strain CCIBT1059 was taken and incubated over ten days in the growth chamber under the following conditions: BG 11m, ph 9.5, temperature 25 ºC, photoperiod light dark cycle and irradiance of µmol photons.m 2.s 1. This adapted culture was used as inoculums for the ph treatments, initiated with 1.5 x 10 4 cells.ml 1. Cells were inoculated into 1l glass balloons with 500 ml of medium BG 11m. The ph was adjusted varying the amount of NaOH 0.1M and HCl 0.1M in the medium. The treatments (n = 3) were the following: ph 7.0, 9.5 and During 30 days, daily analyses in Fuchs Rosenthal counting chamber were performed for determining the growth (cells.ml ¹) and biovolume (mm 3.l 1 ) for every treatment. Growth rates were calculated during the exponential phase and are presented as relative growth rate (µ, day 1 ). Mean doubling time represents the time period necessary to duplicate the cell population (G, day 1 ), both estimated according to Fo g g & Th a k e (1987). The cell yield (R; cells.ml 1 ), corresponding the maximum number of cells subtracted of the cell number of the inoculums, was also calculated. All treatments were analyzed using the analysis of variance (ANOVA) and Tukey test (Program GraphPad Prism version 5.0). Results of all tests were considered significant at 95% confidence if p < 0.05 for a given F statistic test value. Results Morphological characteristics of A. elenkinii The following morphometric parameters, observed during the exponential growth of all treatments (Fig. 2, Supplementary Table 1. electronic version only at fottea.czechphycology.cz), showed significant differences when compared with nature material: lower values of cell length in the treatments, higher values of cell width in the treatments, lower values of cell length/width ratio in the treatments, and higher values of length and width of heterocytes and number of cells in trichomes were also observed in the treatments (p < ). Regarding the number of coils, more tightly coiled trichomes were observed only at ph 10.5 in comparison with nature material (p < ). During the stationary growth (Fig. 2), significant differences were observed in all treatments when compared with nature material in the following parameters: lower values of length and width of cells were observed in all treatments ( p < ) and number of cells in trichome as well as length and width of heterocytes showed higher values in treatments (p < ). Regarding the length/width ratio of cells, significant difference were observed at ph 9.5 (p < ) and 10.5 (p < 0.003) and lower values were observed in these treatments. Furthermore, the length/width ratio of heterocytes higher values were observed at ph 7.0 (p < 0.003) and 9.5 (p < ). As with the exponential growth, more tightly coiled trichomes

3 Fottea 11(1): , Fig. 1. Aspects of alkaline lake and species studied: (a) general view of the lake Salina do Meio photo A. Pott; (b) occurrence of bloom of the Anabaenopsis elenkinii in lake; (c z ) A. elenkinii; (c j) nature material; (k z ) strain CCIBT1059; (k p) ph 10.5 on day 0, 7, 10, 14, 20 and 30 respectively; (q v) ph 9.5 on day 0, 4, 10, 14, 20 and 30 respectively; (w z ) ph 7.0 on day 0, 8, 10, 14, 20 and 30 respectively. Scale bar 10μm.

4 122 Santos et al.: Growth and morphology of Anabaenopsis elenkinii Fig. 2. Box and whisker plots of morphological characteristics of Anabaenopsis elenkinii strain CCIBT1059 in the nature material and exponential and stationary growth at different treatment of ph. Whiskers represent minimum and maximum values, boxes symbolize ± standard deviation, and + inside boxes are mean values. L/w = length/width. (p < ) were observed only at ph 10.5 on stationary growth in comparison with nature material (Fig. 2). According to figure 2, the variability within each treatment between exponential and stationary growth was clearly higher than the variability among different treatments in most cases. Regarding the length of cells, statistical differences were observed only at ph 7.0, in which higher values were encountered at stationary growth. The length of heterocytes showed statistical difference only at ph 10.5 and lower values were observed at

5 Fottea 11(1): , Fig. 3. Growth rate and cell yield of Anabaenopsis elenkinii strain CCIBT1059 on exponential growth at different treatment of ph. The cultures were incubated at 25ºC, irradiance of µmol photons.m 2.s 1 on a hr light dark cycle on modified medium BG 11 (3% NaNO 3 ). Error bars denote the standard deviation (n = 3). Asterisks above the bars indicate significant differences at p < Biovolume mm 3.l -1 l Days Fig. 4. The growth curves (Biovolume mm 3.l 1 ) of Anabaenopsis elenkinii strain CCIBT1059 at different ph treatments. The cultures were incubated at 25ºC, irradiance of 90 µmol photons.m 2.s 1 on a hr light dark cycle on modified medium BG 11 (3% NaNO 3 ). Error bars indicate the standand deviation (n = 3). stationary growth (p < 0.05). However, the width of cells and heterocytes showed lower values at stationary growth in comparison with exponential growth at ph 7.0, 9.5 and The number of cells and coils per trichomes and the length/width ratio of heterocytes showed statistical difference between both phases of growth only at ph 9.5, in which slightly coiled trichomes were observed on exponential growth, whereas a higher number of cells per trichome and heterocytes slightly longer were encountered on stationary growth (Fig. 2, Supplementary Table 2 electronic version only at fottea.czechphycology.cz). In nature material and in all treatments, the coiled trichomes were the dominant form, but in each treatment a small percentage of straight trichomes were observed at stationary growth (Fig. 1 c x). The longest trichomes were found at ph 7 (maximum 45 cells on day 14) in comparison with ph 9.5 (maximum 32 cells on day 10) and

6 124 Santos et al.: Growth and morphology of Anabaenopsis elenkinii ph 10.5 (maximum 23 cells on day 8). Although statistical analyses showed significant difference in the number of cells per trichomes, the real difference in the mean values was slight (ca. 1 cell). Moreover, the average during the cultivation time (30 days) was approximately 8 cells per trichome in all treatments (Suppl. Tables 1, 2). Growth of A. elenkinii The effect of different ph on growth rate (µ; day 1 ), cell yield (R; cells.ml 1 ) and duplication time (G; day 1 ) of the A. elenkinii strain CCIBT1059 are shown in Fig. 3 and Table 1. Higher values of growth rate and cell yield were observed at ph 10.5 and were statistically different from ph 7 (p [µ] < 0.05; p [R] < ) and 9.5 (p [µ] < 0.05; p [R] < ). Consequently, the duplication time of cells was lower at ph 10.5 and higher at ph 7 and 9.5 (Table 1). In ph 7 and in ph 9.5 were not observed statistically difference in relation to growth rate, cell yield and duplication time of cells. Figure 4 shows the growth curves (biovolume mm 3.l 1 ) of A. elenkinii at different treatments of ph. Discussion Morphologically, the nature material of A. elenkinii is similar to that of Anabaenopsis hungarica Halász because of the small number of cells/ trichome (average 4, varying from 2 to 6 cells) and by the cylindrical cells (Fig. 1, c j). However, the Brazilian material frequently presents longer trichomes (up to12 cells) and elliptical cells (Sa n t o s & Sa n t An n a 2010). Besides, the metric parameters are also very distinct: A. hungarica presents 2.5 µm wide trichomes and the A. elenkinii trichomes are wider (4.5 µm) (Ko m á r e k 2005). According to morphometric parameters, the material from Pantanal (from nature and from culture) is similar to A. elenkinii and the variability observed was considered as part of the natural variability of this species (Ko m á r e k 2005, Sa n t o s & Sa n t An n a 2010). The occurrence of terminal and intercalary pairs of heterocytes was observed in all treatments (Fig. 1 k z ), but akinetes were never formed during the cultivation time. Based on our results, the occurrence of heterocytes was proportionally equal to the density of trichomes in all treatments (not shown), indicating that the ph does not affect the heterocyte formation. Akinetes were found only in nature, where they appeared during the dry season of the years 2006 and 2009 (ph and water temperature ºC) and on the rain season of 2006 (ph 10.2 and water temperature 33 ºC) (Santos & Sa n t Anna 2010). The absence of akinetes in the analyzed treatments indicates that the tested culture conditions do not represent stress conditions that induce the production of resistance structures. For this reason, in spite of the lower growth rate at ph 7.0 and 9.5 (Table 1) compared with the literature (growth rates of , according to Mu r et al. 1999), akinetes were never formed. Although the influence of ph on akinetes sporulation has been documented for Anabaenopsis arnoldii Ap t e k a r by Re d d y (1984), our results indicate that ph does not seem to be directly involved in akinetes formation of A. elenkinii from the alkaline lakes of Pantanal. Environmental features such as temperature (Shafik et al. 2003; Mo o r e et al. 2004), phosphorus concentration (Moore et al. 2004) and light intensity (Moore et al. 2005) showed great influence on akinetes formation of Cylindrospermopsis raciborskii (Wo l o s z y ń s k a) Se e n a y y a et Su b b a Ra j u. However, Zapomělová et al. (2008) studied the effects of the temperature, light intensity and nutrient composition on morphological traits of Nostocaceae (Anabaena, Aphanizomenon, Nostoc, Scytonema and Tolypothrix), but did not manage to prove the influence of these parameters on the akinetes formation. In all treatments, A. elenkinii strain CCIBT1059 showed tendency to decrease the cell width at the stationary growth compared with that at the exponential growth (Fig. 2). The same observation was documented to C. raciborskii (Hawkins et al. 2001). The length/width ratio of cells tends to increase during the cultivation time because the cell width decreases independently of ph. According to Ha r r i s (1986), phytoplanktonic cells with high growth rate tend to have a smaller cell volume. In spite of no significant difference on the growth of A. elenkinii between the treatment at ph 7 and that at ph 9.5, variability of the width and length/width ratio of cells was observed. Treatment at ph 9.5 presented shorter cells than that at ph 7.0, both in the exponential growth phase and at the stationary phase (Fig. 2). Another interesting aspect observed was the morphological similarity of the trichomes between the treatment at ph 7.0

7 Fottea 11(1): , and that at ph 10.5 during the exponential growth, except for the number of coils/trichome (Fig. 2). The higher values of growth rate and cell yield observed at ph 10.5 indicate that A. elenkinii is typical of alkaline systems and also that in lower ph values the growth limitation can occur in terms of density and biomass (Table 1). Thus, the bloom formation of A. elenkinii in the alkaline lakes from Brazilian Pantanal is favored by the high ph values towards its high productivity. Acknowledgements The first author thanks the financial supports of Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP Process number 2009/ ). We also thank the Embrapa Pantanal for permission to collect phytoplankton samples in Nhumirim farm, and Dr. Arnaldo Yoso Sakamoto (Universidade Federal de Mato Grosso do Sul UFMS Campus de Três Lagoas) for the logistics support for the collection expedition. References Ba l l o t, A., Da d h e e c h, P.K., Ha a n d e, S. & Kr i e n i t z, L. (2008): Morphological and phylogenetic analysis of Anabaenopsis abijatae and Anabaenopsis elenkinii (Nostocales, Cyanobacteria) from tropical inland water bodies. Microb. Ecol. 55: Ba r b i é r o, L., Qu e i r o z Ne t o, J.P., Ci o r n e i, G., Sa k a m o t o, A.Y., Ca p e l l a r i, B., Fe r n a n d e s, E. & Va l l e s, V. (2002): Geochemistry of water and ground water in the Nhecolândia, Pantanal of Mato Grosso, Brazil: variability and associated processes. Wetland 22: Ca r v a l h o, L.R., Pi p o l e, F., We r n e r, V.R., La u g h i n g h o u s e IV, H.D., Ca m a r g o, A.C.M., Ra n g e l, M., Ko n n o, K. & Sa n t An n a, C.L. (2008): A toxic cyanobacterial bloom in an urban coastal lake, Rio Grande do Sul State, Southern Brazil. Braz. J. Microbiol. 39: Eg b o r g e, A.B.M. (1994): Salinity and the distribution of rotifers in the Lagos Harbour Badagry Creek system, Nigeria. Hydrobiologia 272: Fo g g, G.E. & Th a k e, B. (1987): Algae cultures and phytoplankton ecology, 3 rd ed. 269 pp., Wisconsin, University Wisconsin Press, Madison. Ha r r i s, G.P. (1986): Phytoplankton ecology: structure, function and fluctuation. 384 pp., Chapman and Hall Press, London. Ha r t, B.T., Ba i l e y, P., Ed w a r d s, R., Ho r t l e, K., Ja m e s, K., McMa h o n, A., Me r e d i t h, C. & Sw a d l i n g, K. (1991). A review of the salt sensitivity of the Australian freshwater biota. Hydrobiologia 210: Ha w k i n s, P.R.; Pu t t, E.; Fa l c o n e r, I.; Hu m p a g e, A. (2001): Phenotipical variation in a toxic strain of the phytoplankter, Cylindrospermopsis raciborskii (Nostocales, Cyanophyceae) during batch culture. Environ. Toxicol. 16: Ho f f m a n n, L., Ko m á r e k, J. & Ka š t o v s k ý, J. (2005): System of cyanoprokaryotes cyanobacteria) state in Algological Studies 117: Ju n k, W.J., Cu n h a, C.N., Wa n t z e n, K.M., Pe t e r m a n n, P., St r ü s s m a n n, C., Ma r q u e s, M.I. & Ad i s, J. (2006): Biodiversity and its conservation in the Pantanal of Mato Grosso, Brazil. Aquat. Sci. 68: Ko m á r e k J. (2005). Phenotypic diversity of the heterocytous cyanoprokaryotic genus Anabaenopsis. Czech Phycology 5: Kr i s t j á n s s o n, J.K. & Hr e g g v i d s s o n, G.O. (1995): Ecology and habitats of extremophiles. Word. J. Microbiol. Biotechnol Mo o r e, D., McGr e g o r, G.B. & Gl e n, S. (2004): Morphological changes during akinetes germination in Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria). J. Phycol. 40: Mo o r e, D., O Do n o h u e, M., Ga r n e t t, C., Cr i t c h l e y, C. & Sh a w, G. (2005): Factors affecting akinete differentiation in Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria). Freshwater Biol. 50: Mu r, L.R., Sk u l b e r g, O.M. & Ut k i l e n, H. (1999): Cyanobacteria in the environment. In: Chorus, I. & Bartram, J. (eds): Toxic cyanobacteria in water. A guide to their Public Health Consequences, Monitoring and Management. 416 pp., E & FN Spon, London & New York. Re d d y, P.M. (1984): Influence of ph on sporulation, spore germination and germling survival in blue green algae. Acta Hydrochim. Hydrobiol. 12: Sh a f i k, H.M., Vö r ö s, L., Sp r ó b e r, P., Pr é s i n g, M. & Ko v á c s, A.W. (2003): Some special morphological features of Cylindrospermopsis raciborskii in batch and continuous cultures. Hydrobiologia : Sa n t o s, K.R.S. & Sa n t An n a, C.L. (2010): Cianobactérias de diferentes tipos de lagoas ( salina, salitrada e baía ) representativas do Pantanal da Nhecolândia, MS, Brasil. Revista Brasil. Bot. 33: Si l v a, J.S.V. & Ab d o n, M.M. (1998): Delimitação do Pantanal brasileiro e suas sub regiões. Pesquisa Agropecuária Brasileira 33: Tu d o r a n c e a, C. & Ha r r i n s o n, A.D. (1988): The benthic communities of the saline lakes Abijata

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